RNAi-based therapeutics is in various stages of study for treating a number of different diseases, including viral infections (e.g., hepatitis, HIV, influenza, RSV), cancer, hyperlipidemia, Huntington's disease, and age-related macular degeneration, to name a few. Our previous studies have provided important information on the rate- limiting processes in primary cells in tissues that limit RNAi effectiveness due to toxicity. The first section of the application is designed to further dissect the molecular limitations of RNAi. We propose studies that examine the limitations of RNAi delivery via a transcriptional template or direct double-stranded delivery, while still developing a robust RNAi delivery system that can safely knock down transgene expression by 3 to 4 orders of magnitude. This level of knockdown will likely be important for a number of the diseases under consideration for preclinical study and clinical efficacy. The second part of the application deals with new discoveries that we uncovered while examining the RNAi pathway in mammals. First, we discovered new mechanisms on how microRNAs are loaded into the RISC complex. Second, we found the biologic basis for why miRNA targets in the 3'UTR are heavily favored over those in the coding regions of mRNAs. A third and forth discovery are related to identifying two new classes of small RNAs. One group of these RNAs may be involved in RNA-directed RNA transcription, a process known to be robust in invertebrates and plants but not previously believed to exist in mammals. The second class of small RNAs is derived from tRNAs. We discovered two different types of abundant tRNA- derived small RNAs. We have established in part how these small RNAs are generated from the tRNAs. While we have established that these RNAs are localized to the cytoplasm, our preliminary data suggest that these RNAs are not microRNA-like, even though they bind to specific argonaute proteins. We hypothesize that these RNAs serve as a buffer to regulate the number of miRNAs that can be taken up into an active RISC complex. We propose studies to attempt to further elucidate their function. Moreover, we have discovered that for at least the type II tRNA-derived small RNAs, there is the potential to use them in combination with an antisense oligonucleotide for regulated gene silencing in a process we call Sense-Induced-Trans Silencing or SITS. We propose studies to determine the likelihood of developing this approach into a bona-fide therapeutic.